Enhancing meiotic recombination in wheat by modulating RECQ helicases

Project Outline

Wheat accounts for 20% of the calories and protein consumed by humans and is the largest crop in the UK, but yields have plateaued and are susceptible to decline due to extreme weather conditions. Wheat breeding is a numbers game; the more crosses generated, the greater the chance of generating useful trait combinations. However, breeding is dependent on the frequency and distribution of crossovers (CO) that are few in number (1-3 per chromosome pair) and skewed towards the chromosome ends, so even with a large number of crosses, desired combinations may not be attained. CO initiation sites are not limited by location or number, but only ~2% mature into COs, suggesting potential within the system to increase recombination.

In humans, mutations in the RECQ genes, Bloom’s syndrome helicase (BLM) and Werner’s helicase (WRN) are associated with premature ageing and early onset of cancer. RECQs are conserved throughout eukaryotes and repair DNA by homologous recombination. In Arabidopsis thaliana, the BLM orthologues (RECQ4a/RECQ4b) function redundantly as anti-recombinases during meiosis1. Arabidopsis does not contain a WRN ortholog, but we have identified one in wheat (RECQ7), that is a pro-CO factor2. RECQ4 is an anti-CO factor, so the two proteins may function antagonistically in processing DNA recombination intermediates. In wheat we have isolated knockout mutants of RECQ4 and RECQ7 as well as generating RECQ7 overexpression lines that require analysis for altered recombination patterns. We will use state-of-the-art super-resolution fluorescence microscopy in conjunction with immunolocalisation and a panel of antibodies developed in the lab that target specific proteins in the CO pathway. Using this approach we will be able to determine the spatio-temporal dynamics of recombination protein loading on meiotic chromosomes. Molecular markers will also be utilised to monitor recombination frequency.

There are three outstanding questions that will be investigated in this PhD project:

Q1. How does RECQ7 function as a pro-recombinase in homologous DNA repair?

Q2. Does RECQ4 act as an anti-recombinase during meiosis in wheat?

Q3. Can we determine an antagonistic mechanism of action between RECQ4 and RECQ7 in wheat?

References

Séguéla-Arnaud M, Crismani W, Larchevêque C, Mazel J, Froger N, Choinard S, Lemhemdi A, Macaisne N, Van Leene J, Gevaert K, De Jaeger G, Chelysheva L, Mercier R. (2015) Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM. PNAS;112(15):4713-8.

Gardiner LJ, Wingen LU, Bailey P, Joynson R, Brabbs T, Wright J, Higgins JD, Hall N, Griffiths S, Clavijo BJ, Hall A. (2019) Analysis of the recombination landscape of hexaploid bread wheat reveals genes controlling recombination and gene conversion frequency. Genome Biology 20(1):69.

Techniques

Super-resolution structured illumination microscopy, immunolocalization, fluorescence in situ hybridisation, PCR, cloning, DNA sequencing

Investigating the role of SUMOylation in meiotic recombination and chromosome segregation in Arabidopsis

Project Outline

Post-translational modifications of proteins such as phosphorylation, methylation and acetylation have been extensively studied in eukaryotes and now the Small Ubiquitin modifier (SUMO) is gaining attention for its importance in fundamental biological roles. SUMO is a small protein that can be conjugated to target proteins through a cascade of E1, E2 and E3 ligases. Recently, SUMO has been mapped to yeast meiotic proteins, indicating an essential role in regulating homologous recombination1. We have identified two potential E3 ligases (based on protein structure), that are required for normal recombination to proceed in Arabidopsis thaliana. Interestingly, in the mutant of one of the genes, crossover sites are altered in number and position, indicating that SUMOylation is required for crossover patterning. The aim of this project will be to characterise one of the E3 ligase mutants and determine its function during meiosis.

You will analyse chromosomes of the Arabidopsis mutants with structured illumination super-resolution microscopy in conjunction with a panel of antibodies that we have generated in the lab that specifically bind to meiotic proteins that control crossover number and position. This will give you an insight into the mutant phenotype and what is going wrong in the absence of the E3 ligase. You will also perform yeast-2-hybrid experiments to identify which proteins the E3 ligase binds to and which lysine residues are targeted. Once interacting proteins have been identified you will perform an in vitro analysis to detect if they are mono- or polySUMOylated. If successful, you will mutate SUMOylated lysine residues and transform the Arabidopsis mutants by complementation to determine the functionality of SUMOylation by phenotypical analysis with cytological and genetic techniques. In addition, you will learn molecular techniques such as cloning, PCR, DNA sequencing and protein analysis.

References

Bhagwat NR, Owens SN, Ito M, Boinapalli JV, Poa P, Ditzel A, Kopparapu S, Mahalawat M, Davies OR, Collins SR, Johnson JR, Krogan NJ, Hunter N SUMO is a pervasive regulator of meiosis. (2021) Elife. 10:e57720. doi: 10.7554/eLife.57720.

Techniques

Super-resolution structured illumination microscopy, immunolocalization, fluorescence in situ hybridisation, PCR, cloning, DNA sequencing

Unlocking the genetic potential of barley by modulating recombination

Project Outline

Background

Barley is a major worldwide crop used for malting and animal feed. However, breeding new varieties is constrained by the frequency of genetic crossovers (1-3 crossovers per chromosome pair) and their distribution (biased towards the chromosome ends) that underpin crop improvement1,2. Genetic crossovers form during meiosis, a specialised cell division that is required to halve the number of chromosomes for gamete production. This project aims to utilise the phenotype of barley pachytene checkpoint 2 (pch2)3 CRISPR/Cas mutants (that we have generated) that appear to delay the timing of meiotic recombination, thus enabling crossovers to form in chromosomal regions that rarely or never recombine.

In addition to the wider aim of crop improvement, this project will also investigate fundamental biological questions regarding crossover control in eukaryotes by utilising cutting edge microscopy techniques. This will include structured illumination super-resolution microscopy in conjunction with a panel of antibodies that we have generated in the lab that specifically bind to meiotic proteins that control crossover number and position1,2. By perturbing recombination dynamics in the pch23 mutants we will be able to monitor each step of the recombination pathway and determine which sites are selected for crossover designation. We will also develop KASP molecular markers to genetically map recombination in the mutant and test how this compares to the cytological data. Barley is an excellent model for investigating meiotic recombination in species with large chromosomes when compared to the more extensively studied small chromosomes of Arabidopsis thaliana, thus providing a more complete picture of how these conserved processes adapt and function in sexually reproducing eukaryotes.

Aims and Objectives

Determine crossover positions on wild type and pch2 mutant barley chromosomes utilising ZYP1 and HEI10/MLH3 antibodies

Determine the relationship between crossover position and epigenetics utilising antibodies raised to specific chromatin modifications and DNA methylation

Determine the role of PCH2 and ZYP1 in crossover control by assessing recombination dynamics in the pch2 mutant and ZYP1 RNAi knockdown lines

Determine timing of meiosis in pch2 mutants by incorporating DNA base analogs during premeiotic replication

Develop KASP markers to measure recombination in wild type and pch2 mutant

References

Higgins JD, Perry RM, Barakate A, Ramsay L, Waugh R, Halpin C, Armstrong SJ, Franklin FC. (2012) Spatiotemporal asymmetry of the meiotic program underlies the predominantly distal distribution of meiotic crossovers in barley. Plant Cell. 2012 24(10):4096-109. doi: 10.1105/tpc.112.102483.

Barakate A, Higgins JD, Vivera S, Stephens J, Perry RM, Ramsay L, Colas I, Oakey H, Waugh R, Franklin FC, Armstrong SJ, Halpin C. (2014). The synaptonemal complex protein ZYP1 is required for imposition of meiotic crossovers in barley. Plant Cell. 26(2):729-40. doi: 10.1105/tpc.113.121269.

Lambing C, Osman K, Nuntasoontorn K, West A, Higgins JD, Copenhaver GP, Yang J, Armstrong SJ, Mechtler K, Roitinger E, Franklin FC (2015). Arabidopsis PCH2 Mediates Meiotic Chromosome Remodeling and Maturation of Crossovers. PLoS Genetics, 11(7):e1005372. doi: 10.1371/journal.pgen.1005372.

Techniques

Super-resolution structured illumination microscopy, immunolocalization, fluorescence in situ hybridisation, PCR, cloning, DNA sequencing